Normal human lifespan is marked by a complex series of developmental transitions, relative stability during adulthood, and ultimately a gradual decline in viability. Biological clocks presumably underlie the developmental events that occur through childhood and adolescence, but the nature of those clocks has remained obscure. Progress in this area would be of considerable importance, not only for our understanding of child development, but also because instability in putative clock-like mechanisms may occur as part of the aging process. Such instability could well compromise tissue function and contribute to many of the common degenerative diseases of later life. In previous studies, age-related chromatin remodeling was mapped across a region positioned 11 megabases from the human chromosome 4p terminus (4p16.1). This region, extending over 0.7 megabases, exhibits diminishing histone H4 acetylation over a time interval spanning fetal development to early childhood. A second remodeling domain, less than 3 Mb from the 4q terminus (4q35.2), was found through comparisons between young and old adults. Despite marked time frame differences, chromatin changes in the two regions are broadly similar. These findings are novel and support the hypothesis that clock-like mechanisms can reside in chromatin-based epigenetic structures. The past year has seen two major shifts in research emphasis. First, cells studied earlier were from immortalized cell lines or tissue bank-derived non-immortal lineages. Protocols are now in place to investigate human tissues from several clinical sources. Peripheral blood monocytes are obtained from newborns (cord blood) through a collaboration with the Perinatology Branch, NICHD, while monocytes from adults are available through the NIH Department of Transfusion Medicine. Human skin fibroblasts from newborns and adults have likewise been obtained, the latter under a new NICHD Institutional Review Board (IRB) approved protocol. The establishment of this IRB-approved protocol for human tissue procurement represents an essential step in the important goal of studying developmental and common age-related disease states. Second, emphasis of the current research has shifted from direct screens for chromatin remodeling towards the use of RNA expression microarrays to search for instances of development- and age-related epigenetic change - including both chromatin- and DNA methylation-based mechanisms. It bears note that searches for epigenetic changes via alterations in RNA expression are more indirect, and to some extend of higher risk, than are direct chromatin screens. Nevertheless, insofar as a key goal is to link epigenetic mechanisms to specific genes and clinical disorders, the utilization of RNA expression arrays is likely the most efficacious approach. Substantial progress has been made in implementing these new research directions. The differentiation program that transforms monocytes into antigen-presenting dendritic cells (triggered in response to IL4 and GM-CSF) has revealed evidence of both developmental- and age-related change. Such change is highly selective, strongly affecting only a small number of genes, while global aspects of differentiation remain well preserved. Detailed studies are underway with selected genes to determine to what degree alterations in RNA levels reflect transcriptional or nuclear processing controls, and to what extent expression is heterocellular. Results to date indicate that genes subject to both differentiation and developmental controls often exhibit a high degree of variance in expression levels during developmental or age-related transitions. That this occurs, at least in part, through epigenetic mechanisms is supported by chromatin mapping data. In a general way, the well-studied combination of differentiation and developmental controls on fetal hemoglobin expression may prove to be an important paradigm for the developmental- and age-linked transitions observed in the current project. Concurrent work with newborn and adult (young and old) skin fibroblasts remains at a more preliminary stage than studies using the monocyte-dendritic system. Nonetheless, in preliminary experiments, striking differences have been observed in the responsiveness of cells from these respective sources to serum deprivation and refeeding. A long term advantage of the fibroblast system is that detailed in vitro mechanistic studies should be possible using the wide variety of gene transfer and gene knock-down tools now available.